Single-cell analysis reveals transcriptional dynamics in healthy primary parathyroid tissue.

Studies on human parathyroids are generally limited to hyperfunctioning glands owing to the difficulty in obtaining normal human tissue. We therefore obtained non-human primate (NHP) parathyroids to provide a suitable alternative for sequencing that would bear a close semblance to human organs. Single-cell RNA expression analysis of parathyroids from four healthy adult M. mulatta reveals a continuous trajectory of epithelial cell states. Pseudotime analysis based on transcriptomic signatures suggests a progression from GCM2 hi progenitors to mature parathyroid hormone (PTH)-expressing epithelial cells with increasing core mitochondrial transcript abundance along pseudotime. We sequenced, as a comparator, four histologically characterized hyperfunctioning human parathyroids with varying oxyphil and chief cell abundance and leveraged advanced computational techniques to highlight similarities and differences from non-human primate parathyroid expression dynamics. Predicted cell-cell communication analysis reveals abundant endothelial cell interactions in the parathyroid cell microenvironment in both human and NHP parathyroid glands. We show abundant RARRES2 transcripts in both human adenoma and normal primate parathyroid cells and use coimmunostaining to reveal high levels of RARRES2 protein (also known as chemerin) in PTH-expressing cells, which could indicate that RARRES2 plays an unrecognized role in parathyroid endocrine function. The data obtained are the first single-cell RNA transcriptome to characterize nondiseased parathyroid cell signatures and to show a transcriptomic progression of cell states within normal parathyroid glands, which can be used to better understand parathyroid cell biology.

Multiparameter analysis of timelapse imaging reveals kinetics of megakaryocytic erythroid progenitor clonal expansion and differentiation.

Single-cell assays have enriched our understanding of hematopoiesis and, more generally, stem and progenitor cell biology. However, these single-end-point approaches provide only a static snapshot of the state of a cell. To observe and measure dynamic changes that may instruct cell fate, we developed an approach for examining hematopoietic progenitor fate specification using long-term (> 7-day) single-cell time-lapse imaging for up to 13 generations with in situ fluorescence staining of primary human hematopoietic progenitors followed by algorithm-assisted lineage tracing. We analyzed progenitor cell dynamics, including the division rate, velocity, viability, and probability of lineage commitment at the single-cell level over time. We applied a Markov probabilistic model to predict progenitor division outcome over each generation in culture. We demonstrated the utility of this methodological pipeline by evaluating the effects of the cytokines thrombopoietin and erythropoietin on the dynamics of self-renewal and lineage specification in primary human bipotent megakaryocytic-erythroid progenitors (MEPs). Our data support the hypothesis that thrombopoietin and erythropoietin support the viability and self-renewal of MEPs, but do not affect fate specification. Thus, single-cell tracking of time-lapse imaged colony-forming unit assays provides a robust method for assessing the dynamics of progenitor self-renewal and lineage commitment.

Differentiation of PTH-Expressing Cells From Human Pluripotent Stem Cells.

Differentiation of pluripotent stem cells into functional parathyroid-like cells would accelerate development of important therapeutic options for subjects with parathyroid-related disorders, from the design and screening of novel pharmaceutical agents to the development of durable cellular therapies. We have established a highly reproducible directed differentiation approach leading to PTH-expressing cells from human embryonic stem cells and induced pluripotent stem cells. We accomplished this through the comparison of multiple different basal media, the inclusion of the CDK inhibitor PD0332991 in both definitive endoderm and anterior foregut endoderm stages, and a 2-stage pharyngeal endoderm series. This is the first protocol to reproducibly establish PTH-expressing cells from human pluripotent stem cells and represents a first step toward the development of functional parathyroid cells with broad applicability for medicinal and scientific investigation.

Effect of a matrigel sandwich on endodermal differentiation of human embryonic stem cells.

Definitive endoderm can be derived from human embryonic stem cells using low serum medium with cytokines involved in the epithelial-to-mesenchymal transition, including Activin A and Wnt3A. The purpose of this study was to develop an improved protocol that permits the induction of definitive endoderm while avoiding the high rate of cell death that often occurs with existing protocols. By including insulin and other nutrients, we demonstrate that cell viability can be preserved throughout differentiation. In addition, modifying a matrigel sandwich method previously reported to induce precardiac mesoderm allows for enhanced endodermal differentiation based on expression of endoderm-associated genes. The morphological and migratory characteristics of cells cultured by the technique, as well as gene expression patterns, indicate that the protocol can emulate key events in gastrulation towards the induction of definitive endoderm.

Genomic imprinting of IGF2 in marsupials is methylation dependent.

BACKGROUND: Parent-specific methylation of specific CpG residues is critical to imprinting in eutherian mammals, but its importance to imprinting in marsupials and, thus, the evolutionary origins of the imprinting mechanism have been the subject of controversy. This has been particularly true for the imprinted Insulin-like Growth Factor II (IGF2), a key regulator of embryonic growth in vertebrates and a focal point of the selective forces leading to genomic imprinting. The presence of the essential imprinting effector, DNMT3L, in marsupial genomes and the demonstration of a differentially methylated region (DMR) in the retrotransposon-derived imprinted gene, PEG10, in tammar wallaby argue for a role for methylation in imprinting, but several studies have found no evidence of parent-specific methylation at other imprinted loci in marsupials. RESULTS: We performed the most extensive search to date for allele-specific patterns of CpG methylation within CpG isochores or CpG enriched segments across a 22 kilobase region surrounding the IGF2 gene in the South American opossum Monodelphis domestica. We identified a previously unknown 5'-untranslated exon for opossum IGF2, which is flanked by sequences defining a putative neonatal promoter, a DMR and an active Matrix Attachment Region (MAR). Demethylation of this DMR in opossum neonatal fibroblasts results in abherrant biallelic expression of IGF2. CONCLUSION: The demonstration of a DMR and an active MAR in the 5' flank of opossum IGF2 mirrors the regulatory features of the 5' flank of Igf2 in mice. However, demethylation induced activation of the maternal allele of IGF2 in opossum differs from the demethylation induced repression of the paternal Igf2 allele in mice. While it can now be concluded that parent-specific DNA methylation is an epigentic mark common to Marsupialia and Eutheria, the molecular mechanisms of transcriptional silencing at imprinted loci have clearly evolved along independent trajectories.

Ancient and continuing Darwinian selection on insulin-like growth factor II in placental fishes.

Despite abundant examples of both adaptation at the level of phenotype and Darwinian selection at the level of genes, correlations between these two processes are notoriously difficult to identify. Positive Darwinian selection on genes is most easily discerned in cases of genetic conflict, when antagonistic evolutionary processes such as a Red Queen race drive the rate of nonsynonymous substitution above the neutral mutation rate. Genomic imprinting in mammals is thought to be the product of antagonistic evolution coincident with evolution of the placenta, but imprinted loci lack evidence of positive selection likely because of the ancient origin of viviparity in mammals. To determine whether genetic conflict is a general feature of adaptation to placental reproduction, we performed comparative evolutionary analyses of the insulin-like growth factor II (IGF2) gene in teleost fishes. Our analysis included several members of the order Cyprinodontiformes, in which livebearing and placentation have evolved several times independently. We found that IGF2 is subject to positive Darwinian selection coincident with the evolution of placentation in fishes, with particularly strong selection among lineages that have evolved placentation recently. Positive selection is also detected along ancient lineages of placental livebearing fishes, suggesting that selection on IGF2 function is ongoing in placental species. Our observations provide a rare example of natural selection acting in synchrony at the phenotypic and molecular level. These results also constitute the first direct evidence of parent-offspring conflict driving gene evolution.

Allelic expression of IGF2 in live-bearing, matrotrophic fishes.

The parental conflict, or kinship, theory of genomic imprinting predicts that parent-specific gene expression may evolve in species in which parental investment in developing offspring is unequal. This theory explains many aspects of parent-of-origin transcriptional silencing of embryonic growth regulatory genes in mammals, but it has not been tested in any other live-bearing, placental animals. A major embryonic growth promoting gene with conserved function in all vertebrates is insulin-like growth factor 2 (IGF2). This gene is imprinted in both eutherians and marsupials, as are several genes that modulate IGF2 activity. We have tested for parent-of-origin influences on developmental expression of IGF2 in two poeciliid fish species, Heterandria formosa and Poeciliopsis prolifica, that have evolved placentation independently. We found IGF2 to be expressed bi-allelically throughout embryonic development in both species.